Influenza is probably the most wide-spread virus disease in the human population worldwide. Epidemics, which occur annually in most locations, cause 3-5 million severely ill patients per year worldwide. Of these, 250,000 to 500,000 have a fatal outcome. Influenza disease is also the major cause of lost work- and schooldays, which means an economic loss estimated at 87 billion dollars for the USA alone. Both influenza A and the very distantly related influenza B strains cause severe epidemics, the latter at a frequency of about 33%.
Pandemics, which emerge on average once per decade, are even deadlier, because no lingering immune memory is left in the human population. The “Spanish Flu” which spread at the end of World War I, caused an estimated 50 million victims. Also the more recent “Bird flu” was very deadly.
Hemagglutinin (HA) is the most prevalent protein on the viral capsid, and its function is to anchor the virus on its target cells. Serological evidence first suggested that epidemics arose by a few mutations in the HA gene of a circulating strain leading to partial or complete resistance to humoral immunity in the host. New pandemic strains, on the other hand, arise by reassortment, whereby a totally new HA gene (e.g. H1, H2, H3, H5, . . . ) is incorporated in a human influenza strain. These mutations and reassortments may occur in humans or, more likely, in transmissible carrier-animals, such as chickens and swine. The source of these new HA genes is usually migratory water-fowl. Their northern habitat is a well-known as a reservoir of various influenza stains. As a result, the origin of new human pandemics has often been traced to such migratory bird routes. The gradual mutations leading to new epidemics are referred to as “drift”, while the appearance of a new HA gene in a human influenza strain is known as a “shift”. Sequencing of HA genes of appropriate influenza strains confirmed the molecular explanation of the “drift” and “shift” phenomena.
Around 1950, the first influenza vaccines became available. These contain HA as their main antigenic component, either as inactivated whole virus, or dissociated viral capsid proteins or purified subunits. Also, attenuated viral strains were introduced. The result was and still is that these vaccines are highly virus strain specific. Each year in February, the WHO guesses which strains will likely be prevalent that year, and advises a vaccine composition consisting of two influenza A strains and one (recently even two) influenza B strains to the vaccine manufacturers. These classical vaccines, based on induction of anti-HA antibodies, protect very efficaciously provided the guess was right and the HA in the vaccine matches closely the HA in the epidemic viral strain. Because of the uncertainty of strain selection and the nearly annual change of prevalent virus challenge, the vaccination coverage is usually not very high.
The molecular nature of the HA-gene in a newly emerging pandemic strain cannot be predicted. As soon as a new virus appears in humans, its HA-gene is characterized, and a matching vaccine is prepared, tested and distributed. However, as the time to prepare a matching vaccine is on average 6 months, the influenza pandemic may already have caused considerable damage in the human population before it is possible to contain it.
The Influenza M2 protein is a proton-selective ion-channel protein, integral in the viral envelope of the influenza A virus. The channel itself is a homotetramer (i.e. it consists of four identical M2 units), where the units are helices stabilized by two disulfide bonds. It is activated by low pH. The M2 protein is also an integral membrane protein expressed on the surface of infected cells.
The M2 protein has an important role in the infectious cycle of the influenza A virus. It is located in the viral envelope. It allows the entry of protons into the viral particle (virion) from inside the endosome, thus lowering the pH inside of the virus. This causes dissociation of the viral matrix protein M1 from the ribonucleoprotein RNP; this dissociates the nucleic acid genome and the membrane, so when fusion occurs, the nucleic acid is released into the cytoplasm and not stick to the membrane, where it cannot carry out its function.
The function of the M2 channel can be inhibited by the antiviral drugs amantadine and rimantadine, which then blocks the virus from taking over the host cell. The molecule of the drug binds to the transmembrane region, sterically blocking the channel. This stops the protons from entering the virion, which then does not disintegrate. However, when one of five amino acids in the transmembrane region gets suitably substituted, the virus gains resistance to the existing M2 inhibitors. As these mutations are relatively frequent, presence of the selection factors (e.g. using amantadine for treatment of sick poultry) can lead to emergence of a resistant strain.
The amino acid sequence of the M2 protein can vary between different virus strains. One example (M2 Influenza A/Puerto Rico 8/34) is represented by SEQ ID NO: 89.
Antibodies against the M2 protein are known in the art and commercially available. They are applicable e.g. in Western Blot, immunohistochemistry, ELISA or enzyme immunoassay. These antibodies comprise polyclonal and monoclonal antibodies generated in animal hosts.
Common treatments for an influenza infection include a range of medications and therapies. They may either directly target the virus itself or may just offer relief to symptoms. The two main classes of antiviral drugs used against influenza are neuraminidase inhibitors (such as zanamivir and oseltamivir) and inhibitors of the viral M2 protein, such as amantadine and rimandatine. These drugs can reduce the severity of symptoms if taken soon after infection and can also be taken to decrease the risk of infection. Influenza viruses can show resistance to anti-viral drugs, which can result from over-use of these drugs.
In the case of neuraminidase inhibitors, different strains of influenza viruses have differing degrees of resistance against these antivirals, and it is impossible to predict what degree of resistance a future pandemic strain might have.
The M2 inhibitors are sometimes effective against influenza A if given early in the infection. In the case of amantadine, the treatment may lead to rapid production of resistant viruses, and over-use of this drug has probably contributed to the spread of resistance. Measured resistance to amantadine and rimantadine in American isolates of H3N2 has increased to 91% in 2005.
There remains hence a need for further options for the treatment of influenza. Accordingly, there is provided herewith means and methods for the solution of this problem in the form of an antibody construct comprising a first binding domain specific for the extracellular part of the influenza M2 protein and a second binding domain specific for CD3.